Treatment of liver disease with orthotopic liver transplantation (OLT) carries considerable morbidity and mortality. Moreover, due to organ shortages, thousands of people die each year without getting transplanted. Therefore, safer and more convenient alternative therapies will benefit many people requiring liver transplantation. An approach that might address this problem is the development of a proliferative cell line that expresses liver-specific genes which could be employed for cell transplantation or for a bioartificial liver. Developing such a line from human embryonic stem cells (hESC) would also provide cells valuable for pharmacology and toxicology studies. Specific Aims: 1) to determine conditions for directing the hESC to differentiate into hepatocytes in vitro;2) to characterize the differentiated hESC;3) to establish the in vivo potential of hESC in NOD-SCID mice;and 4) to establish the in vivo potential or hESC in nonhuman primates. Methods: A variety of conditions will be empirically assayed to delineate the most effective approach to differentiate the hESC along a hepatocyte lineage. This will include empiric studies of optimizing media, extracellular matrix, and growth factors. The purity of the cells will be enhanced with transduction of liver-specific lent virus vectors. The purified cells will be characterized with assays of liver-specific gene products, growth factor responsiveness, and potential oncogenicity. To determine whether the cells can engraft, proliferate, and function after transplantation, studies will be conducted in immunodeficient NOD- SCID mouse models of liver cell injury and in nonhuman primates. Innovative imaging approaches will be utilized to assess the viability and proliferation of the cells over time in vivo. Health Relatedness: If the studies are successfully undertaken, it will provide for the development of an unlimited source of differentiated human hepatocytes that can be used for toxicology and pharmacology studies, and can be employed in cell-based therapeutics in man.